RESUMO
Antigen 2 is a glycosylated protein present in the cell walls of the dimorphic fungus Coccidioides immitis. Using oligodeoxyribonucleotide (oligo) primers based on the sequences of Ag2 cDNA, the gene encoding Ag2 was cloned from genomic DNA derived from the mycelial phase of C. immitis by PCR. Nucleotide (nt) sequence analyses showed a 582 base pair (bp) ORF disrupted by two introns which are 78 bp and 101 bp long. The deduced primary translation product consists of 194 amino acids (aa), contains an N-terminal putative signal sequence to allow transport into the endoplasmic reticulum, and a C-terminal putative signal sequence to enable a GPI anchor addition. Putative GPI anchor/cleavage site and O-glycosylation sites, as well as phosphorylation and myristoylation sites are also present. On the basis of these analyses, we predict that a prepro-Ag2 undergoes a post-translational modification to yield the mature glycosylated Ag2 protein which is anchored on the extracellular plasma membrane of mycelial and spherule-phase cells.
Assuntos
Antígenos de Fungos/imunologia , Coccidioides/imunologia , Proteínas Fúngicas/imunologia , Glicoproteínas/imunologia , Sequência de Aminoácidos , Antígenos de Fungos/química , Sequência de Bases , Coccidioides/química , DNA Fúngico , Proteínas Fúngicas/química , Glicoproteínas/química , Dados de Sequência MolecularRESUMO
Neither the presence nor the specific role of secretory cytokines in in vivo allograft rejection has been extensively studied. We quantitated the levels of colony-stimulating factors, tumor necrosis factor, and interleukin 1 within the rejecting allograft. BALB/c (H-2d) mice were implanted with polyurethane sponges containing either allogeneic C57BL/6 (H-2b) or syngeneic splenocytes, or splenocyte-free media. At various days postgrafting, the sponges were harvested, and the cells infiltrating the grafts were analyzed for specific antidonor cytolytic activity, while IL-1, TNF, and CSF levels were measured in the graft exudate fluid. Allogeneic grafts had significantly higher concentrations of CSF, TNF, and IL-1 than syngeneic of splenocyte-free grafts. A specific radioimmunoassay revealed that macrophage colony-stimulating factor (M-CSF) is the primary CSF produced in the grafts. Peak TNF levels preceded peak M-CSF and IL-1 levels, which coincided with the initial appearance of allospecific cytotoxic T lymphocytes. Maximal CTL activity was seen on day 13, when the levels of these cytokines had already begun to fall. Specific bioassays for multi-CSF (IL-3), granulocyte CSF, granulocyte-macrophage CSF, IL-2, and IL-4 failed to detect these cytokines in the sponge fluid at any time. We hypothesize that TNF, M-CSF, and IL-1 probably play regulatory roles in the immunologic events at the site of allograft challenge.
Assuntos
Fatores Estimuladores de Colônias/biossíntese , Interleucina-1/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Animais , Fatores Estimuladores de Colônias/análise , Feminino , Fator Estimulador de Colônias de Granulócitos , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Substâncias de Crescimento/análise , Fator Estimulador de Colônias de Macrófagos , Camundongos , Camundongos Endogâmicos , Poliuretanos , Radioimunoensaio , Baço/citologia , Baço/imunologia , Baço/transplante , Tampões de Gaze Cirúrgicos , Transplante Homólogo/imunologiaRESUMO
Although different populations of inflammatory cells infiltrate the healing wound, the mechanisms by which they influence the healing process in vivo are poorly defined. In vitro studies suggest that these cells may mediate wound healing by releasing various cytokines within the wound. We measured the levels of interleukin (IL) 1, IL-2, IL-3, IL-4, IL-6, tumor necrosis factor, and macrophage colony-stimulating factor within a subcutaneously implanted polyurethane sponge on various days after injury. Significantly higher levels of IL-1, IL-6, tumor necrosis factor, and macrophage colony-stimulating factor were detected in the wound fluid compared with basal serum levels in nonwounded mice. Tumor necrosis factor, macrophage colony-stimulating factor, and IL-6 peaked earlier than IL-1; however, the levels of these cytokines had fallen by the 13th day after wounding. Interleukin 2, IL-3, and IL-4 could not be detected in the wound fluid, and the wound fluid inhibited the proliferation of the IL-2-dependent cell lines CTLL-2 and HT-2 in response to recombinant IL-2. We hypothesize that tumor necrosis factor, macrophage colony-stimulating factor, IL-1, and IL-6, which are secreted at the site of injury, interact to promote tissue remodeling. The decrease in the levels of these cytokines by the 13th day after wounding may be the result of a regulatory process by the healed wound.
Assuntos
Fatores Biológicos/biossíntese , Cicatrização , Animais , Ensaio de Unidades Formadoras de Colônias , Fatores Estimuladores de Colônias/biossíntese , Citocinas , Feminino , Interleucina-1/biossíntese , Interleucina-2/biossíntese , Interleucina-4/biossíntese , Interleucina-6/biossíntese , Fator Estimulador de Colônias de Macrófagos , Camundongos , Camundongos Endogâmicos BALB C , Poliuretanos , Fator de Necrose Tumoral alfa/biossíntese , Ferimentos e Lesões/etiologia , Ferimentos e Lesões/metabolismoRESUMO
In this series of experiments we have shown that there are at least two mechanisms of CSF production by spleen cells in vitro: nonspecific secretion of CSF by nonimmune cells and immune, antigen-driven production by specific T-lymphocytes. M-CSF was mainly produced in the nonspecific reaction. The immune production by T-lymphocytes consisted of increased CSF production and secretion of multiple species of CSFs.
Assuntos
Fatores Estimuladores de Colônias/biossíntese , Listeriose/imunologia , Baço/imunologia , Animais , Ensaio de Unidades Formadoras de Colônias , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Substâncias de Crescimento/biossíntese , Listeriose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Salmonella typhimurium/imunologia , Baço/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
We investigated production of colony-stimulating factors by Listeria monocytogenes-immune spleen cells. Levels of total colony-stimulating factors in supernatants from antigen-stimulated immune cells were increased two- to fourfold over those in supernatants from nonimmune cells. Immune supernatants primarily induced formation of granulocyte colonies, whereas nonimmune supernatants induced formation of macrophage colonies. Immune supernatants had two- to 10-fold higher levels of macrophage colony-stimulating factor, as determined by radioimmunoassay, and higher levels of interleukin-3 and possibly granulocyte-macrophage colony-stimulating factor, as determined by factor-dependent cell line growth, than did nonimmune supernatants. Using enrichment and depletion techniques we showed that L3T4-positive T lymphocytes were responsible for most of the colony-stimulating factor production in the immune reaction.
Assuntos
Antígenos de Bactérias/imunologia , Fatores Estimuladores de Colônias/biossíntese , Listeriose/imunologia , Linfócitos T/imunologia , Animais , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Granulócitos , Substâncias de Crescimento/biossíntese , Imunização , Interleucina-3/biossíntese , Listeria monocytogenes/imunologia , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Radioimunoensaio , Baço/imunologia , Linfócitos T/metabolismoRESUMO
Murine T lymphocyte clones sensitized to Listeria monocytogenes were developed to investigate specific mechanisms of T cell-mediated immunity. The clones were of the Thy-1.2+, L3T4+, Lyt-2- phenotype and proliferated in a dose response fashion to heat-killed Listeria. Cloned T lymphocytes injected intravenously protected nonimmune mice against L. monocytogenes challenge as determined by spleen and liver bacterial numbers. Supernatants, produced by stimulating clones with heat-killed Listeria for 48 h, also afforded protection against L. monocytogenes. The clonal supernatants contained significant quantities of IFN-gamma and CSF. IFN-gamma production was Ag specific and occurred within 24 h of stimulation. CSF production by clones was increased four- to sixfold over baseline as determined by a bone marrow colony-forming assay and was Ag specific. When the IFN-gamma in supernatants was neutralized with a specific mAb, protection afforded by the supernatants was lost. These data indicate that one mechanism for Ag-specific, T lymphocyte-mediated protection against L. monocytogenes is the release of IFN-gamma.
Assuntos
Antígenos de Diferenciação de Linfócitos T , Interferon gama/biossíntese , Listeriose/prevenção & controle , Linfócitos T/transplante , Animais , Antígenos de Bactérias/imunologia , Sistema Livre de Células , Células Clonais/classificação , Células Clonais/metabolismo , Células Clonais/transplante , Fatores Estimuladores de Colônias/metabolismo , Epitopos/imunologia , Feminino , Imunização Passiva , Listeriose/imunologia , Listeriose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Testes de Neutralização , Fenótipo , Linfócitos T/classificação , Linfócitos T/metabolismoRESUMO
CSF have been postulated to be important mediators of host defenses. The current studies were undertaken to investigate the production of CSF by Listeria-specific, T cell clones and to assess the participation of CSF in anti-listerial host resistance. Listeria-specific L3T4+, Lyt-2- T cell clones were isolated and expanded by standard techniques. The clones themselves protected mice from listerial challenge when injected intravenously, and supernatants generated from Ag-stimulated clones were protective. In order to define factors important in the protection, supernatants from the clones were assayed for CSF by several in vitro assays. Total colony-stimulating activity was measured with a bone marrow colony-forming assay. T cell clones secreted 1000 to 2000 U/ml of colony-stimulating activity after 48 hours of stimulation with specific antigen. The relative amounts of the various CSF were determined by the capacity of supernatants to support proliferation of the factor-dependent cell lines FDCP-1 and 32D cl 3 in the presence and absence of specific anti-CSF antibodies. Results showed that most of the CSF activity was due to granulocyte-macrophage (GM)-CSF and IL-3. The role of GM-CSF in anti-listerial host resistance was assessed in two types of experiments. In one set of experiments GM-CSF activity was neutralized in the supernatants by addition of specific rabbit anti-GM-CSF antibodies. Treated and untreated supernatants were then tested for their capacity to protect nonimmune mice against listerial challenge. Neutralization of GM-CSF in the supernatants decreased the protective capacity of the supernatants by approximately 23%. In a second set of studies, the administration of recombinant murine GM-CSF was shown to protect mice from challenges of L. monocytogenes. Taken together, these experiments provide evidence that CSF are important mediators of immune T cell mediated host defenses.
Assuntos
Fatores Estimuladores de Colônias/biossíntese , Imunização Passiva , Linfócitos T/metabolismo , Animais , Linhagem Celular , Sistema Livre de Células , Células Clonais/imunologia , Células Clonais/metabolismo , Células Clonais/transplante , Fatores Estimuladores de Colônias/imunologia , Fatores Estimuladores de Colônias/fisiologia , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Substâncias de Crescimento/imunologia , Soros Imunes/farmacologia , Imunidade Inata , Listeriose/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Testes de Neutralização , Coelhos , Linfócitos T/imunologia , Linfócitos T/transplanteRESUMO
The proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha), and interleukin-6 (IL-6) were induced in mice infected with Coccidioides immitis. Analyses of the cytokine profiles of two inbred mouse strains which differ in their susceptibility to pulmonary challenge with C. immitis revealed higher levels of IL-6 in lungs from DBA/2 mice (resistant strain) than in those from BALB/c mice (susceptible strain) beginning at day 6 and continuing through day 15 postinfection. Spleen cells from both mouse strains secreted TNF-alpha, IL-1 alpha, and IL-6 in vitro in response to stimulation with killed spherules but differed in that spleen cells from the resistant strain produced increased levels of these cytokines earlier after pulmonary challenge and at increased levels throughout the course of the disease.
Assuntos
Coccidioidomicose/imunologia , Interleucina-1/biossíntese , Interleucina-6/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Especificidade da EspécieRESUMO
The profiles of gamma interferon (IFN-gamma) and interleukin-4 (IL-4) production were evaluated during the course of coccidioidomycosis in two inbred mouse strains which differ in their susceptibility to Coccidioides immitis. Cytokine responses, measured at the molecular and protein levels, showed increased levels of IFN-gamma in lung extracts from mice of the resistant DBA/2 strain after a pulmonary challenge, whereas the susceptible BALB/c strain manifested a predominant IL-4 response. The importance of these cytokines in host defense against C. immitis was established by treating the mice with recombinant cytokines or neutralizing anticytokine monoclonal antibodies. Treatment of the susceptible BALB/c mice with recombinant murine IFN-gamma significantly protected mice against systemic challenge, and in the reciprocal experiment, the administration of an anti-IFN-gamma monoclonal antibody to the resistant DBA/2 mice significantly decreased their capacity to control disease. Although the treatment of DBA/2 mice with recombinant IL-4 did not alter the disease, neutralization of endogenous IL-4 in infected BALB/c mice by administration of a neutralizing anti-IL-4 antibody led to a significant reduction in the fungal load in their tissues. These results, taken together, establish that IFN-gamma plays a pivotal role in resistance to C. immitis, whereas IL-4 down-regulates protective immunity against C. immitis.
Assuntos
Coccidioidomicose/imunologia , Interferon gama/fisiologia , Interleucina-4/fisiologia , Animais , Sequência de Bases , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Proteínas Recombinantes/farmacologiaRESUMO
We have previously reported on the alternate regulation of gamma interferon (IFN-gamma) and interleukin-4 (IL-4) in inbred mouse strains which differ in their susceptibility to Coccidioides immitis. The genetically resistant DBA/2 mice manifest a predominant T-helper 1 (Th1) response, with early production of IFN-gamma, whereas susceptible BALB/c mice show an early production of the Th2 cytokine IL-4. Since IL-12 is one cytokine that can act early during host defenses to promote the differentiation of cytokine production towards IFN-gamma and thus may promote expression of a protective immune response, we investigated the role of IL-12 in resistance to C. immitis. Administration of recombinant IL-12 to the susceptible mouse strain before and after systemic (intraperitoneal) challenge with C. immitis significantly ameliorated the course of the disease, as measured by a reduction in the fungal load in the lungs, liver, and spleen. Analysis of the cytokine mRNA in lungs from infected BALB/c mice revealed that the protective effect of recombinant IL-12 was accompanied by a shift from a Th2 to a Th1 response. The importance of IL-12 in resistance to this fungus was further established by showing that neutralization of endogenous IL-12 in the resistant DBA/2 mouse strain led to a significant increase in the fungal burden in pulmonary and extrapulmonary tissues. These results establish that IL-12 plays a pivotal role in the host defense against systemic challenge with C. immitis.
Assuntos
Coccidioides/imunologia , Coccidioidomicose/imunologia , Interleucina-12/fisiologia , Animais , Coccidioidomicose/terapia , Feminino , Imunoterapia , Interleucina-12/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Proteínas Recombinantes , Células Th1/imunologia , Células Th2/imunologiaRESUMO
Experiments were performed to determine the effect of starvation on T-cell mediated host defences. In mice starved for 72 hr, the number of thymocytes fell by 98%, spleen cells by 82% and peripheral blood cells by 44%. By 7 days after the end of starvation, values had returned to within 50% of baseline. The percentage of L3T4 and Lyt-2 antigen-bearing cells fell in the thymus, but the percentage of Thy-1.2-positive cells did not change. Starvation decreased the percentage of lymphocytes in peripheral blood but increased the percentage of granulocytes. During starvation, the cellularity in thymuses, spleens and peripheral blood was preserved in adrenalectomized mice compared to normal or sham-adrenalectomized mice. Confirming previous results of ours, starved mice were resistant to i.v. challenge with Listeria monocytogenes immediately after starvation. However, when starved mice were immunized with a sublethal dose of Listeria immediately after starvation and challenged 3-4 weeks later, they were less resistant to Listeria than fed, immunized mice. Similarly, spleen cells of starved, immunized mice had a reduced capacity to transfer immunity passively to non-immune mice. Increasing the immunizing dose of Listeria in starved mice increased the level of immunity that developed. These data indicate that starved mice have a marked reduction in T-cell cellularity, possibly related to corticosteroid production during the stress of starvation. Although starved mice were relatively resistant to Listeria immediately after starvation, they had a reduced capacity to develop T-cell mediated immunity to Listeria. This deficiency could be partly overcome by increasing the immunizing dose of Listeria.
Assuntos
Inanição/imunologia , Linfócitos T/imunologia , Doença Aguda , Glândulas Suprarrenais/fisiopatologia , Animais , Antígenos de Superfície/análise , Contagem de Células , Feminino , Imunidade Celular , Imunização Passiva , Listeriose/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Baço/imunologia , Timo/imunologiaRESUMO
Experiments were performed to elucidate the role of colony-stimulating factors in host defenses to the intracellular pathogen Listeria monocytogenes. Mice were protected against Listeria sp. by adoptive transfer of immune spleen cells and were then challenged with listeriae intravenously. Control mice were injected with spleen cells from uninfected mice. Adoptively immunized (immune) mice had significantly fewer listeriae in spleens and livers 2 and 4 days after Listeria challenge than did control mice. During acute infection, colony-stimulating activity in serum was increased earlier (10 h) in immune mice than in controls. Concentrations of colony-stimulating activity were equal at 24 h. By 48 h, values were decreased in immune mice, but were elevated in control mice. Similar changes were noted when a specific colony-stimulating factor, macrophage colony-stimulating factor, was measured in serum by using a radioimmunoassay. The changes in serum colony-stimulating activity in mice adoptively immunized with immune spleen cells were eliminated if spleen cells were first treated with anti-Thy-1.2 monoclonal antibodies. The number of macrophage progenitor cells in bone marrow and spleen were also determined as measures of the hemopoietic potential in these organs. The number of macrophage progenitor cells in bone marrow was higher in immune animals than control animals at 1, 2, and 4 days of infection. Similarly, the number of these cells in spleens was higher during the early stages of infection in immune mice. These results indicate that both the regulation of leukocyte production and the transfer of specific cellular immunity by spleen cells are associated, and they therefore suggest that hemopoietic regulatory factors play a role in immune host defenses.
Assuntos
Fatores Estimuladores de Colônias/fisiologia , Listeriose/imunologia , Macrófagos/fisiologia , Animais , Antígenos de Superfície/análise , Células da Medula Óssea , Ensaio de Unidades Formadoras de Colônias , Células-Tronco Hematopoéticas/fisiologia , Imunidade , Imunização Passiva , Listeria monocytogenes/imunologia , Camundongos , Baço/citologia , Baço/imunologia , Antígenos Thy-1RESUMO
We have previously cloned the cDNA fragment that encodes the complement fixation antigen of Coccidioides immitis. The recombinant protein was highly sensitive in detecting CF antibody in sera from patients with coccidioidomycosis but was not specific to C. immitis, as evidenced by its reactivity with sera from patients with histoplasmosis and, to lesser extent, blastomycosis. We undertook this study to determine if the epitope(s) that reacts with CF antibody is the same or differs from the epitopes that are shared with Histoplasma capsulatum and Blastomyces dermatitidis. PCR-generated CF/chitinase cDNA fragments were cloned and examined for their reactivity in enzyme-linked immunosorbent assays using sera from patients with coccidioidomycosis, histoplasmosis, or blastomycosis. A peptide domain comprised of amino acid residues 20 through 310 was shown to express an epitope(s) that is specific to anti-Coccidioides CF antibody. The peptide detected serum antibody in 21 (95%) of 22 patients with active coccidioidomycosis and was without reactivity with sera from 20 patients with histoplasmosis, 15 patients with blastomycosis, and 14 healthy subjects. Antibody titers to the recombinant peptide directly correlated with CF antibody titers (P < 0.01), and preadsorption of reference CF antiserum with the peptide ablated the reactivity of the antiserum in the immunodiffusion assay for CF antibody. The delineation of a recombinant peptide that has both sensitivity and specificity will provide a valuable tool for detecting CF antibody and for evaluating the role of CF antibody in the host response to C. immitis.
Assuntos
Anticorpos Antifúngicos/sangue , Antígenos de Fungos/imunologia , Coccidioidomicose/imunologia , Testes de Fixação de Complemento , Mapeamento de Epitopos , Linfócitos B/imunologia , Blastomicose/imunologia , Coccidioidomicose/sangue , Coccidioidomicose/patologia , Reações Cruzadas , DNA Complementar/genética , Ensaio de Imunoadsorção Enzimática , Biblioteca Gênica , Histoplasmose/imunologia , Humanos , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Interleukin 12 (IL-12) plays an important role in the induction of protective immunity against cancer and infectious diseases. In this study we asked whether IL-12 cDNA could increase the protective capacity of the antigen 2 (Ag2) gene vaccine in experimental coccidioidomycosis. Coimmunization of BALB/c mice with a single-chain IL-12 cDNA (p40-L-p35) and Ag2 cDNA, both subcloned into the pVR1012 plasmid, significantly enhanced protection against systemic challenge with 2,500 arthroconidia, as evidenced by a greater-than-1.3-log-unit reduction in the fungal load in the lungs and spleens compared to mice receiving the pVR1012 vector alone, Ag2 cDNA alone, or IL-12 cDNA alone. The enhanced protection was associated with increased gamma interferon secretion; production of immunoglobulin G2a (IgG2a), IgG2b, and IgG3 antibodies to Coccidioides immitis antigen; and the influx of CD4(+) and CD8(+) T cells in lungs and spleens. When challenged by the pulmonary route, mice covaccinated with Ag2 cDNA and IL-12 cDNA were not protected at the lung level but did show a significant reduction in the fungal load in their livers and spleens compared to mice vaccinated with Ag2 cDNA or IL-12 cDNA alone. These results suggest that IL-12 acts as a therapeutic adjuvant to enhance Ag2 cDNA-induced protective immunity against experimental coccidioidomycosis through the induction of Th1-associated immune responses.
Assuntos
Antígenos de Fungos/genética , Coccidioides/imunologia , DNA Complementar/imunologia , Vacinas Fúngicas/imunologia , Glicoproteínas/genética , Interleucina-12/genética , Vacinas de DNA/imunologia , Animais , Anticorpos Antifúngicos/sangue , Feminino , Proteínas Fúngicas , Glicoproteínas/imunologia , Imunoglobulina G/sangue , Interferon gama/biossíntese , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , PlasmídeosRESUMO
T-cell-mediated immunity is an important determinant in protection against primary infection with Coccidioides immitis, a dimorphic fungal pathogen that causes the disease coccidioidomycosis. To determine if interleukin-12 (IL-12) gene therapy could potentiate host response against C. immitis, we constructed a single-chain cDNA encoding the p40 and p35 subunits linked by a polylinker and, using a retroviral vector, transfected J774 macrophages with the construct. The transduced J774 cells expressed IL-12 in vitro, with a mean concentration of 28,440 pg from 10(6) cells in 48 h as measured by an IL-12 (p75)-specific enzyme-linked immunosorbent assay. The secreted IL-12 was biologically active, as judged by its ability to induce the production of gamma interferon (IFN-gamma) by spleen cells from BALB/c mice. Treatment of the highly susceptible BALB/c mouse strain with the IL-12-transduced J774 cells inhibited C. immitis growth in tissues from mice challenged by a pulmonary route, as evidenced by 1.37-, 2.59-, and 1.22-log reductions in the number of CFU in the lungs, spleens, and livers, respectively, compared to the fungal load in mice given vector-transduced J774 cells. The protective effect of IL-12 gene therapy was accompanied by increased levels of IFN-gamma in the lungs and sera of mice treated with IL-12-transduced J774 cells and the constitutive production of IFN-gamma by their spleen cells cultured in vitro. These results suggest that IL-12 gene therapy could be used as adjunct therapy for coccidioidomycosis.
Assuntos
Coccidioidomicose/terapia , Terapia Genética/métodos , Vetores Genéticos , Interleucina-12/genética , Retroviridae , Células 3T3 , Administração Intranasal , Animais , Coccidioidomicose/imunologia , Citocinas/genética , Feminino , Expressão Gênica , Interferon gama/biossíntese , Interleucina-12/imunologia , Interleucina-12/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Células Th1/imunologiaRESUMO
Tumor necrosis factor-alpha was detected in supernatants collected from BALB/c mouse peritoneal macrophages incubated continuously with Histoplasma capsulatum. The levels of TNF alpha measured by actinomycin D bioassay peaked within hours after exposure and then greatly declined by 24 h. TNF alpha was also measured in bronchoalveolar lavage fluid from BALB/c mice challenged intranasally with H. capsulatum. Lavage fluid TNF alpha levels exhibited the same pattern as the in vitro supernatants; they peaked within hours after challenge and lower levels were detected at 24 h. Treatment of mice with anti-TNF alpha antibody accelerated mortality in response to systemic infection and significantly increased tissue colony counts in the liver and spleen. In the murine model, TNF alpha is produced in response to H. capsulatum and appears to play some role in host defense to infection.
Assuntos
Histoplasma/imunologia , Histoplasmose/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Líquido da Lavagem Broncoalveolar/química , Células Cultivadas , Contagem de Colônia Microbiana , Vida Livre de Germes , Imunidade Celular , Fígado/microbiologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Baço/microbiologia , Fator de Necrose Tumoral alfa/análiseRESUMO
Host defense against murine Chlamydia trachomatis (mouse pneumonitis agent [MoPn]) in a murine model was investigated. Gamma interferon (IFN-gamma) was produced in the lungs by both MoPn-susceptible nude athymic (nu/nu) and MoPn-resistant heterozygous (nu/+) mice. In vivo depletion of IFN-gamma in nu/nu mice led to exacerbation of infection. Fluorescence-activated cell sorter analysis disclosed induction of GL3 antibody-positive cells (putatively gamma/delta+ T cells) in nu/nu mouse lung during infection with MoPn. Treatment of nu/nu mice in vivo with antibody to NK cells (anti-asialo GM1 antibody) or to gamma/delta cells (UC7-13D5) did not significantly decrease IFN-gamma production in the lung. However, treatment of severe combined immunodeficiency mice (which lack gamma/delta cells) with antibody to NK cells significantly reduced lung IFN-gamma levels.
Assuntos
Infecções por Chlamydia/imunologia , Chlamydia trachomatis , Interferon gama/biossíntese , Pneumonia/imunologia , Animais , Feminino , Células Matadoras Naturais/imunologia , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Receptores de Antígenos de Linfócitos T gama-delta/análise , Linfócitos T/imunologiaRESUMO
We have reported that a murine Histoplasma capsulatum-reactive CD4+ T-cell line and clones thereof did not adoptively transfer protection against H. capsulatum infection in normal or cyclophosphamide-treated C57BL/6 mice. One explanation for the results was that the T cells failed to traffic to lymphoid organs in these animals. In this study, we have sought to determine whether one of these clones, 2.3H3, could mediate protection in nude (C57BL/10) or irradiated (5 Gy) heterozygous nude (nu/+) C57BL/6 mice. Mice were inoculated intravenously with 10(7) resting 2.3H3 cells or with an equal number of cells of the ovalbumin-reactive clone 1S6; 2 h later, the mice were challenged intranasally with 5 x 10(6) yeast cells. By day 5 of infection, lungs, livers, and spleens of nude and irradiated nu/+ mice given 2.3H3 contained significantly fewer (P < 0.05) CFU than the same organs from mice inoculated with 1S6. This effect was specific for H. capsulatum, since 2.3H3 did not reduce the number of Coccidioides immitis CFU in lungs, livers, and spleens of irradiated nu/+ mice. By day 10, the amounts of H. capsulatum CFU in lungs, livers, or spleens of nude and irradiated nu/+ mice inoculated with 2.3H3 were smaller than those in 1S6-inoculated mice, but these differences did not reach statistical significance (P > 0.05). The mortality rate of mice inoculated with 2.3H3 and that of mice inoculated with 1S6 were similar. Histopathological examination of tissues from 2.3H3- and 1S6-inoculated mice demonstrated the presence of granulomatous inflammation in organs from both groups. Tissues from 2.3H3-treated mice contained fewer yeasts per high-power field than tissues from 1S6-treated mice. Thus, irradiated or nude mice are permissive for the expression of protective immunity by a CD4+ T-cell clone. Although the protective capacity of T cells in these animals is transient, these animals will be useful for differentiating protective from nonprotective T-cell clones.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Histoplasmose/imunologia , Imunoterapia Adotiva , Animais , Células Clonais , Feminino , Granuloma/patologia , Histoplasma/isolamento & purificação , Histoplasmose/patologia , Fígado/microbiologia , Fígado/patologia , Pulmão/microbiologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Antigen 2 (Ag2) has been implicated as a T-cell-reactive component of the pathogenic fungus Coccidioides immitis. We report the production of a murine monoclonal antibody (MAb) of the immunoglobulin G2a isotype that recognizes an epitope specific to C. immitis Ag2. This specificity was evidenced by the finding that the MAb did not recognize other antigens present in coccidioidin or spherulin and did not show reactivity with antigenic extracts from Histoplasma capsulatum or Blastomyces dermatitidis. The epitope was labile to enzymatic digestion with pronase but resistant to treatment with glycolytic enzymes and to periodate oxidation. This peptide epitope appears to require conformational structure on the basis that it was not recognized by the MAb in immunoblots of antigen that had been electrophoresed in polyacrylamide gels under denaturing, reducing conditions. Immunoaffinity chromatography of spherulin on columns containing the MAb established that the MAb was effective as a ligand for isolating Ag2 from heterogeneous extracts. The production of a MAb which recognizes an Ag2-specific epitope and its utility as a ligand for isolating Ag2 will provide a valuable reagent for studies of this immunologically important antigen.
Assuntos
Anticorpos Antifúngicos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Fungos/imunologia , Coccidioides/imunologia , Proteínas Fúngicas/imunologia , Animais , Antígenos de Fungos/química , Eletroforese em Gel Bidimensional , Epitopos , Proteínas Fúngicas/química , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The role of CD8+ T cells in antichlamydial immunity was investigated in a murine model of chlamydial genital infection by using T-cell clones generated against the Chlamydia trachomatis agent of mouse pneumonitis (MoPn). Two CD8+ T-cell clones tested (2.1F and 2.14-9) were chlamydia antigen specific and MHC restricted and reacted against MoPn as well as the Chlamydia psittaci agent of guinea pig inclusion conjunctivitis and C. trachomatis serovar E, suggesting the recognition of a genus-specific antigen. Upon adoptive transfer into persistently MoPn-infected nu/nu mice, 55.6% of the recipients of clone 2.1F (15 of 27) resolved the infection but recipients of clone 2.14-9 did not. The ability to resolve the MoPn infection correlated with the capacity of clone 2.1F to elaborate a combination of gamma interferon and tumor necrosis factor alpha. The results suggested that in addition to CD4+ T cells, CD8+ T cells may also contribute to antichlamydial T-cell immunity in vivo.